It is known that heart disease or atherosclerosis is a result of the build up and subsequent restriction of blood vessels by plaque of circulating blood to the heart muscle. It is believed a preemptive factor to plaque development is depositing or loading of the cells comprising the blood vessel lining with cholesterol in the form of cholesteryl ester. It is also believed the cholesteryl ester originates from sources that include lipoproteins, specifically the low density (LDL) and very low density (VLDL) lipoproteins. The high density lipoproteins (HDL), however, are known to be protective elements where heart disease or atherosclerosis is concerned and are desirable lipoproteins.
The medical profession utilizes several methods to minimize the exposure of cells that line the circulatory system to VLDL and LDL with the rationale that lowering the exposure of arterial walls to LDL and VLDL will lower the ability of the cells to load or uptake cholesteryl ester from these lipoproteins. Means of lowering LDL and VLDL include indirect means such as disruption of the cholesterol biosynthetic pathway that occurs within the liver cells. Drugs, such as the statins, including Mevacor® and Lipitor® inhibit HMG-Coenzyme A reductase, a key enzyme along the cellular pathway for production of cholesterol.
Cholesterol is a life sustaining component necessary to maintain many basic functions such as cell membrane integrity. Cholesterol is transported throughout the circulatory system within lipoproteins in the ester form as cholesteryl ester. The ester is completely water insoluble and resides in the core of the lipoproteins. Statin drugs are given in limited doses so that the pathway is not totally without output of cholesterol but the output is partially reduced. The reduced cholesterol output from the liver results in generally a reduction of all cholesterol pools or stores. Included is the reduction of the lipoprotein cholesterol pool. Lipid transfer proteins or other proteins that utilize lipids as substrates interact with the lipoproteins to access their substrates both at the core and the surface of the lipoprotein. One lipid transfer protein, in particular a neutral lipid transfer protein, cholesteryl ester transfer protein (CETP), which shuttles cholesteryl esters from HDL to VLDL and LDL.
There are various known techniques to measure cholesteryl ester transfer protein (CETP) activity. For example, an article entitled: Effect of Very Low-Density Lipoproteins on Lipid Transfer in Incubated Serum, by A. V. Nichols and L. Smith, J. Lipid Research, vol. 6, pp. 206-210 (1965), measures the activity of CETP by determination of cholesteryl ester (CE) mass transfer. The determination of CE mass transfer from high density lipoprotein to very-low density lipoprotein (VLDL) and low density lipoprotein (LDL) requires the re-isolation of VLDL and LDL after incubation with HDL and the CETP source in order to determine the cholesteryl ester mass transfer.
The VLDL/LDL re-isolation from the incubation mixture is a technique that includes ultra-centrifugation of the incubation mixture for many hours so that the VLDL and LDL components are floated upwards through a density gradient as the HDL component of the incubation mixture sinks to the bottom of the centrifuge tube. Further processing of the sample requires a method of determining the amount or mass of cholesteryl ester associated with the re-isolated VLDL or LDL and equating a change in mass to CETP facilitated transfer. Later variations of this method of activity measurement have simplified mass determination by utilizing HDL that has a radioactive label associated with the CE.
An article entitled: Cholesteryl Ester Exchange Protein in Human Plasma Isolation and Characterization by N. M. Pattnaik, A. Montes, L. B. Hughes and D. B. Zilversmit, Biochemica et Biophysica Acta 530, pp. 428-438 (1978), discloses a method of activity measurement of CETP that also utilizes radioactive CE in HDL. This method is an improvement over the above method by simplifying the incubation mixture components separation or the re-isolation technique discussed above. In the cited article, separation of the LDL component from the HDL component is accomplished by precipitation of the LDL component of the incubation mixture. The LDL precipitate is pelleted by a relatively short slow-speed centrifugation and the remaining HDL supernatant is counted. The loss of radioactivity from the HDL component is attributed to 3H-CE transferred to the LDL pellet.
An article titled: Fluorescent Determination of Cholesteryl Ester Transfer Protein (CETP) Activity in Plasma by N. Dousset, L. Douste-Blazy in Clinical Chemistry, vol. 38, No. 2, p. 306 (1982), is an improvement over previous methods of activity measurement since it discloses a technique that does not require radioactive components. In this method, transfer activity of the CETP is determined by the measurement of transfer of a fluorescent labeled CE. In this reference, the cholesteryl ester molecule utilized as the CETP substrate for transfer has been covalently bound to a fluorescent molecule derived from pyrene. The pyrene labeled cholesteryl ester (PY-CE) is recognized by the CETP and the PY-CE may be detected by a fluorimeter. The accumulation of the PY-CE in the LDL fraction is, however, only able to be determined after the separation of the LDL acceptor from the HDL donor.
An article entitled: Enhancement of The Human Plasma Lipid Transfer Protein Reaction by Apolipoproteins by T. G. Milner, K. W. S. Ko, T. Ohnishi, and S. Yokoyama in Biochimica Biophysica Acta 1082, pp. 71-78 (1991), discloses a method for determining the activity of CETP also utilizing a pyrene labeled CE (PY-CE). This method does not require separation or re-isolation of substrates, but uses the measurement of both monomer and excimer fluorescent emission from the pyrene label to determine a ratio thereof. The cited article improves upon certain aspects of the previous method.
An article entitled: Use of Fluorescent Cholesteryl Ester Microemulsions in Cholesteryl Ester Transfer Protein Assays by Charles L. Bisgaier, Laura Minton, Arnold D. Essenberg, Andrew White, and Reynold Homen published in the Journal of Lipid Research, Volume 34, 1993 discloses a method that utilizes a self-quenching fluorescent labeled cholesteryl ester core.
Additionally, there are methods disclosed by the present inventor in U.S. Pat. Nos. 5,770,355; 5,618,683; 5,585,235 and 6,174,693.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.